Prostate cancer is the largest cause of cancer death among men in the United States. Prostate cancer cells produce a variety of autocrine growth factors, among them the parathyroid hormone- related peptide (PTHrP). The first aim of this project will use two approaches to examine the effects of PTHrP on prostate cell growth: establishing cell lines over- or under-expressing PTHrP, and treating cells with recombinant PTHrP (1-139). Thus we can distinguish between the autocrine/paracrine (cell surface) vs. intracrine (nuclear) effects of the peptide. Vitamin D decreases prostate cancer cell growth and epidemiological studies have shown that vitamin D deficiency is a risk factor for prostate cancer. The long-term objective of our research is to determine whether vitamin D or its analogues are useful as chemopreventative or chemotherapeutic agents in prostate cancer. The PTHrP gene is down-regulated by 1,25-dihydroxyvitamin D (1,25(OH)2D), the hormonally active form of vitamin D, in prostate cancer cells. Therefore, vitamin D not only has antiproliferative effects, but decreases PTHrP secretion in prostate cells. This project seeks to define the mechanism(s) by which 1,25(OH)2D, alone and in combination with 9-cis-retinoic acid, represses PTHrP gene expression in normal and cancerous prostate cell lines. The human PTHrP gene is a complex transcriptional unit with at least three different promoters. Different cell lines and tissues exhibit different promoter utilization patterns. The responsiveness of each promoter to 1,25(OH)2D will be assessed using transient transfection assays. The precise sequence elements conferring responsiveness to 1,25(OH)2D within a particular promoter will be located by deletion mapping. Trans-acting nuclear proteins interacting with these promoter elements will be characterized by gel retardation and nuclease protection assays. The hypercalcemic effects of 1,25(OH)2D itself have thus far prevented its prophylactic and therapeutic use, but various non-hypercalcemic vitamin D analogues have recently been synthesized. Some of these analogues will be tested for their ability to down-regulate PTHrP gene expression and decrease PTHrP secretion, thereby addressing whether these derivatives exert the same net desirable effects as the parent compound. These studies should thus provide a molecular basis for use of non-hypercalcemic vitamin D analogues as chemopreventive and chemotherapeutic agents targeted at both PTHrP-mediated prostate cancer cell proliferation and humoral hypercalcemia of malignancy.